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PCR of TCR/Ig gene rearrangements is considered the method of choice for minimal residual disease (MRD) quantification in BCP-ALL, but flow cytometry analysis of leukemia-associated immunophenotypes (FCM-MRD) is faster and biologically more informative. FCM-MRD performed in 18 laboratories across seven countries was used for risk stratification of 1487 patients with BCP-ALL enrolled in the NOPHO ALL2008 protocol. When no informative FCM-marker was available, risk stratification was based on real-time quantitative PCR. An informative FCM-marker was found in 96.2% and only two patients (0.14%) had non-informative FCM and non-informative PCR-markers. The overall 5-year event-free survival was 86.1% with a cumulative incidence of relapse (CIR5y) of 9.5%. FCM-MRD levels on days 15 (HzR 4.0, p p p 5y adjusted for day 29 FCM-MRD, with higher levels in adults (median 2.4 × 10−2 versus 5.2 × 10−3, p 5y = 3.2%). For patients who did not undergo transplantation, day 79 FCM-MRD > 10−4 associated with a CIR5y = 22.1%. In conclusion, FCM-MRD performed in a multicenter setting is a clinically useful method for MRD-based treatment stratification in BCP-ALL.

The percentage of blasts cells in the bone marrow (BM) of MDS patients is one of the key parameters for MDS classification and for the differential diagnosis with acute myeloid leukemia (AML). Currently, the gold standard to determine the blast percentage is conventional cytomorphology. To assess the possible impact of blast cell enumeration in BM biopsies from MDS patients on the final WHO classification using CD34 immunohistochemistry (IHC) a total of 156 BM samples from MDS and MDS-AML patients were studied and compared to blast counts by cytomorphology (CM). Eighty-nine BM aspirates were also studied by flow cytometry (FCM). Percentages of CD34+ blasts by IHC were determined blindly by two hematopathologists. Automated CD34-cell count was performed in 25 cases. Good overall agreement was found for CM and FCM with respect to critical blast thresholds (5%, 10%, 20%) (p p

5-Azacitidine (AZA) therapy is used in high-risk myelodysplastic syndrome (MDS) patients who often show abnormalities in their immunophenotype. We explored the potential impact of AZA on these immunophenotypic abnormalities in serial bone marrow studies performed in 81 patients from five centers. We compared the immunophenotypic features before and after therapy with AZA, established definitions consistent with flow cytometry immunophenotyping (FCI) improvement, and explored its clinical significance. After a median of 6 cycles of AZA, 41% of patients showed a FCI improvement and this finding associated with best possible clinical response (P < 0.001). FCI improvement also correlated with hematological improvement (HI) (53/78 patients; 68%), independently of their eligibility for stem cell transplantation. Among patients who achieved a HI after 6 cycles of AZA, the probability of maintaining this response at 12 cycles of AZA was twice as large (67%) for those patients who also achieved a FCI improvement after 6 cycles of AZA as compared to patients who did not (33%, P < 0.01). These findings support that monitoring of the immunophenotypic abnormalities during therapy with AZA may assist in redefining the quality of response in patients with MDS.

Mixed phenotype acute leukemias (MPALs) represent a rare subgroup of acute leukemias with a poor prognosis. Proper diagnosis and classification of MPAL is extremely important for patients' outcome. Morphology and flow cytometry recognize two types of MPAL: the "bilineal" MPAL with the coexistence of two blast populations of different lineage and truly "biphenotypic" MPAL coexpressing markers of more than one lineage in a homogenous blast population, respectively. The WHO 2008 classification further delineated three categories: associated with t(9;22)/BCR-ABL1 fusion gene, associated with KMT2A (mixed lineage leukemia) rearrangements, and nonotherwise specified. These categories remained unchanged in the WHO2016 update. Molecular studies have further underlined the heterogeneity of MPAL. In this review, rules for the correct assignment of acute leukemia to the MPAL category are discussed, including both flow cytometry and immunohistochemistry on bone marrow or other tissues biopsies. Comparison of the immunophenotypic classification proposals is provided outlining the explorations mandatory for definitive diagnosis. An extensive review of published data summarizes the reported cytogenetic and molecular anomalies. New developments in the understanding of the early stages of hematopoiesis provide clues to the possible etiopathology of these diseases. Finally, current treatment recommendations are summarized and referenced for clinical use, pointing out that allogeneic hematopoietic stem cell transplantation at an early stage should be considered (at least in adult patients). © 2019 International Clinical Cytometry Society.

Objectives: The aim of this study was to determine risk factors for development of acute myeloid leukemia/myelodysplastic syndromes (AML/MDS) in patients with multiple myeloma (MM). Methods: We identified all patients diagnosed with MM in Sweden from January 1st, 1958 to December 31, 2011. A total of 26 627 patients were diagnosed with MM with during the study period. Of these, 124 patients (0.5%) developed subsequent AML/MDS. For each patient with MM and a subsequent AML/MDS diagnosis, we randomly selected a matched (age, sex, and date of MM diagnosis) MM patient without a subsequent second malignancy diagnosis. Results: The cumulative melphalan exposure was significantly higher (OR = 2.8, 95% CI 1.7-5.2; P

Background: Acute promyelocytic leukemia (APL) is one of the most life-threatening hematological emergencies and requires a prompt correct diagnosis by cytomorphology and flow cytometry (FCM) with later confirmation by cytogenetics/molecular genetics. However, nucleophosmin 1 muted acute myeloid leukemia (NPM1+ AML) can mimic APL, especially the hypogranular variant of APL. Our study aimed to develop a novel, Radar plot-based FCM strategy to distinguish APLs and NPM1+ AMLs quickly and accurately. Method: Diagnostic samples from 52 APL and 32 NPM1+ AMLs patients were analyzed by a 3-tube panel of 10-color FCM. Radar plots combining all markers were constructed for each tube. Percentages of positive leukemic cells and mean fluorescence intensity were calculated for all the markers. Results: APL showed significantly higher expression of CD64, CD2, and CD13, whereas more leukemic cells were positive for CD11b, CD11c, CD15, CD36, and HLA-DR in NPM1+ AMLs. Radar plots featured CD2 expression, a lack of a monocytic component, lack of expression of HLA-DR and CD15, and a lack of a prominent CD11c+ population as recurring characteristics of APL. The presence of blasts with low SSC, presence of at least some monocytes, some expression of HLA-DR and/or CD15, and a prominent CD11c population were recurrent characteristics of NPM1+ AMLs. Radar plot analysis could confidently separate all hypergranular APL cases from any NPM1+ AML and in 90% of cases between variant APL and blastic NPM1+ AML. Conclusion: Radar plots can potentially add to differential diagnostics as they exhibit characteristic patterns distinguishing APL and different types of NPM1+ AMLs. (Less)

Background: Myelodysplastic syndromes (MDS) are considered clonal diseases and are diagnosed according to WHO by cytomorphology and cytogenetics. The diagnostic potential of flow cytometric aberrancies has not yet been comprehensively evaluated. Aim: Multicenter prospective evaluation of diagnostic potential of flow cytometric aberrancies predefined according to European LeukemiaNet (ELN). Methods: 1682 patients undergoing diagnostics for suspected MDS according to WHO 2016 criteria were analyzed in parallel by flow cytometry according to ELN recommendations. Results: Median age was 72 years (18-97). MDS, MPN-RS-T or CMML were confirmed by cytomophology in 1029 (61%) cases, 653 (39%) were non-MDS. IPSS-R data was available in 857 (51%). An overall flow cytometric readout was available in 1679 (99.8%). 1001 (60%) were in agreement with MDS while 678 (40%) were not. Flow cytometric readout significantly correlated with cytomorphologic diagnosis (p Non-MDS cases had a fewer myeloid progenitor cells (MPC) (mean±SD, 0.8±0.9%) compared to low-risk MDS (1.7±2.3%, p3% was strongly associated with MDS/CMML (286/293, 98%, p Neutrophil aberrancies were found more frequently in neoplastic cases than in non-MDS cases (table 1). Again, frequencies of aberrations were higher for high-risk MDS as compared to low-risk MDS while this was not the case for CMML showing frequencies rather similar to low-risk MDS. Frequencies of aberrancies in monocytes revealed a similar figure as in neutrophils with higher rates in neoplastic cases but clearly significant numbers positive in non-MDS cases. Interestingly, frequencies were not higher in high-risk MDS as compared to low-risk MDS. As anticipated, frequencies were highest in CMML (table 1). Regarding erythroid cells only an aberrant percentage of them and aberrant CD71 expression were found in a reasonable number of cases. Importantly, rates of positivity were rather high in non-MDS cases which did not differ from CMML cases (table 1). In order to identify the diagnostic value of each individual aberrancy multivariate analyses were performed in the three subgroups, low-risk MDS, high-risk MDS and CMML, as well as in the total cohort. In low-risk MDS ten aberrancies were independently related to MDS (table 2). Five of these aberrancies were found in MPCs, two each in neutrophils and monocytes and one in erythroid cells. In high-risk MDS 11 aberrancies were independently related to MDS (table 2). Eight were found in MPCs, two in neutrophils, none in monocytes and one in erythroid cells. In CMML 12 aberrancies were independently related to CMML (table 2). Four were found in MPCs, neutrophils and monocytes, respectively, and none in erythroid cells. Considering all these three groups together and all aberrancies identified significantly related to MDS/CMML in at least one group in univariate analysis, multivariate analysis identified 12 aberrancies independently related to MDS/CMML (table 2). Six were found in MPCs, two in neutrophils, three in monocytes and one in erythroid cells. Taking into consideration only aberrancies independently associated with MDS/CMML, three such aberrancies resulted in an 80% agreement with the cytomorphologic diagnosis of MDS/CMML, i.e. 20% concordantly negative and 60% concordantly positive. Importantly, this applies without need of at least two cell compartments being affected as specified in the ELN recommendations. Conclusions: This multicenter prospective evaluation confirms the diagnostic potential of flow cytometric aberrancies. A core set of 17 markers identified as independently related to a diagnosis of MDS/CMML is suggested mandatory for flow cytometric evaluation of suspected MDS. An MPC count >3% should be considered indicative of MDS/CMML. Figure 1 Figure 1. Disclosures Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: Equity Ownership. Van de Loosdrecht: Celgene: Consultancy, Research Funding; Amgen: Consultancy; Roche: Consultancy; Novartis: Consultancy; Alexion: Consultancy.

Background Awareness of second malignancies in patients with multiple myeloma (MM) has been increasing during recent years. We have previously shown that second malignancies are associated with a decreased life expectancy in MM patients. Information regarding prior and second malignancies in MM is limited as these patients are often excluded from clinical trials and previously published results from other groups have been conflicting. In the present study we aimed to evaluate two hypotheses. Firstly we hypothesize that prior malignancy is a proxy for genetic instability that could be a risk factor for subsequent malignancy development in MM patients. There is limited data regarding this association in the literature and in two recent registry studies the results were inconclusive. Secondly, to further assess the clinical implication of prior malignancies in MM patients we assessed survival in these patients compared to MM patients without a history of prior malignancy. Patients and Methods All patients diagnosed with MM from January 1, 1973 to December 31, 2013 were identified from the Swedish Cancer Register. All prior and subsequent malignant diagnoses were identified through cross-linkage within the registry. A Cox regression model was used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) where prior malignancy was compared in MM patients who developed a subsequent malignancy and MM patients who did not. In another Cox regression model, survival was compared in MM patients with and without a prior malignancy. The same method was used to estimate if there was a dose-dependent relationship, i.e. if an increasing number of prior malignancies was associated with a poorer outcome. Results A total of 22,359 patients were diagnosed with MM during the study period. Of these, 2,620 (12%) patients had one or more prior malignancy diagnosis at the time of MM diagnosis and 1,243 (6%) patients developed subsequent malignancies. Among the MM patients who developed a subsequent malignancy, 148 (12%) had a prior malignancy diagnosis. Hematological malignancies were 7% of prior malignancies and 17% of subsequent malignancies. MM patients with a prior malignancy diagnosis did not have increased risk of developing a subsequent malignancy compared to MM patients without a prior malignancy (HR 1.0, 95% CI 0.9-1.2). MM patients with a prior malignancy diagnosis had a statistically significant 10% increased risk of death (HR=1.1, 95% CI 1.1-1.2, p Summary and Conclusions In our large population-based study we found that prior malignancy negatively impacts survival in MM patients and that more than one prior malignancy decreases survival even further. Interestingly, a prior malignancy did not increase the risk of developing a subsequent malignancy in MM patients. We confirmed prior reports of solid tumors being more common than hematological malignancies, both prior and subsequent to the MM diagnosis. A prior malignancy was associated with a reduced survival in MM patients without being a risk factor for subsequent malignancies. The underlying explanation for this is probably multifactorial, and could include reduced dose intensity of chemotherapy, complications from treatment, or that MM that develops after another malignancy might be biologically different. Given the increase of cancer survivors in general, our findings are of importance both for the individual patients and their families as well as for the treating physician. Figure Survival in MM patients without a prior cancer diagnosis compared to MM patients with one and two or more prior cancer diagnoses Figure. Survival in MM patients without a prior cancer diagnosis compared to MM patients with one and two or more prior cancer diagnoses Disclosures Landgren: Takeda: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Medscape: Employment, Other: Chairman for Medscape Myeloma Program; Amgen: Speakers Bureau; Celgene: Speakers Bureau; BMS: Speakers Bureau.

We have developed a lymphoproliferative disorder screening tube (LPD-ST) with the aim to provide comprehensive immunophenotyping of lymphocyte subsets with minimal need for additional testing. The LPD-ST consists of CD4/kappa FITC, CD8/lambda PE, CD3/CD14ECD, CD38PC5.5, CD20/CD56PC7, CD10APC, CD19APC-A700, CD5APC-A750, CD57/CD23PB and CD45KO. The LPD-ST was validated against previously used lymphocyte subset panels in Canada (n=60) and in Sweden (n=43) and against the OneFlow™ LST (n=60). The LPD-ST panel was then implemented in clinical practice using dried monoclonal antibody reagents (Duraclone® ) on 649 patient samples in Sweden. In 204 of 649 samples (31%), a monotypic B-cell population was found. Of these cases, a final diagnosis could be rendered in 106 cases (52%), and in the remainder, additional B-cell immunophenotyping was performed. In 20 (3%) samples, an aberrant T-cell population was confirmed by additional testing. Of 425 samples diagnosed as normal/reactive lymphoid tissue, 50 (12%) required additional immunophenotyping, mostly due to an abnormal CD4/CD8 ratio. The LPD-ST tube significantly minimizes the need for additional testing, improves the turn-around time, and reduces the cost of LPD immunophenotyping. It is also suitable for investigating paucicellular samples such as cerebrospinal fluid or fine needle aspirates.

Background: We have evaluated the frequency of lymphoproliferative disorders with more than one aberrant population of monotypic B-cells detected during routine hematopathological diagnostics. Materials and Methods: 2600 samples peripheral (blood, bone marrow, fine-needle aspirate, lymph node, and pleural fluid cell suspensions) were analyzed using a 10-color B-cell panel and a 10-color T-cell panel. A 10-color plasma cell/lymphoplasmacytic panel was performed when appropriate. Results: 790/2600 samples (30%) showed at least one aberrant B-cell population and 27(1%) showed an aberrant T-cell population. 41/790 samples (5.1%) showed two aberrant B-cell populations. Thirteen patients had two B-cell populations with different surface immunoglobulin restriction (one kappa+ and one lambda+), most with B-cell chronic lymphocytic leukemia-related phenotype. Five cases showed two B-cell populations with the same light chain restriction but distinctly different immunophenotypes. In 23 cases, two populations had the same light chain restriction and differed by expression of one or 2 markers, thus, a possibility of intraclonal differentiation could not be excluded. Cases with possible intraclonal differentiation had a significantly higher proportion of aberrant B-cells than those with two coexisting aberrant B-cell populations (49.9% vs. 27.7%, p = 0.008). In only one sample one population of clonal B-cell and one clonal T-cell population with large granular lymphocyte related phenotype were found. Conclusion: Using our panels 5.1% of cases with lymphoproliferative disorder-associated aberrant findings show two aberrant (clonal) lymphoid and/or plasma cell populations. (Less)

Small B cell lymphoid neoplasms are the most common lymphoproliferative disorders involving peripheral blood (PB) and bone marrow (BM). The Bone Marrow Workshop (BMW) organized by the European Bone Marrow Working Group (EBMWG) of the European Association for Haematopathology (EAHP) during the XVIIth EAHP Meeting in Istanbul, October 2014, was dedicated to discussion of cases illustrating how the recent advances in immunophenotyping, molecular techniques and cytogenetics provide better understanding and classification of these entities. Submitted cases were grouped into following categories: (i) cases illustrating diagnostic difficulties in chronic lymphocytic leukaemia (CLL); (ii) cases of BM manifestations of small B cell lymphoid neoplasms other than CLL; (iii) transformation of small B cell lymphoid neoplasms in the BM; and (iv) multiclonality and composite lymphomas in the BM. This report summarizes presented cases and conclusions of the BMW and provides practical recommendations for classification of the BM manifestations of small B cell lymphoid neoplasms based on the current state of knowledge.

Few studies describe the comprehensive immunophenotypic pattern of blastic plasmacytoid dendritic cell neoplasm (BPDCN) in the bone marrow and its treatment. This retrospective analysis evaluates the diagnostic flow cytometry (FCM) pattern and outcome of nine patients diagnosed with BPDCN. A four-tube 10-color FCM panel used for diagnosis of acute leukemia (AL), showed cells in the blast gate (CD45dim/low SSC) and were positive for CD4(bright), CD33(dim), CD56(heterogenous), CD123(bright), CD36, CD38, HLA-DR, CD71. Seven patients received front-line induction therapy with HyperCVAD with an overall response rate of 86%. Five of six responders underwent planned allogeneic hematopoietic cell transplantation (allo-HCT). For a median follow up of 13.3 months, the 1-year disease free survival and overall survival were 56 and 67%, respectively. An accurate diagnosis of BPDCN can be made by 10-color FCM using a four-tube AL panel demonstrating a characteristic pattern of antigen expression. Front-line induction chemotherapy with HyperCVAD can yield high remission rates, but allo-HCT is required for long-term durable remissions.

This review focuses on the most recent literature concerning flow cytometry (FCM) application for diagnosis of myelodysplastic syndrome (MDS). Aberrant FCM results have been defined as abnormalities in at least three tested features comprising at least two bone marrow (BM) cell compartments. FCM results should be interpreted together with the BM smear cytology, the morphological assessment of BM biopsy, and cytogenetic results. Including FCM in the pre-treatment assessment may provide not only diagnostic but also prognostic information. Further studies are needed to evaluate the role of FCM in individual risk assessment for MDS patients and in therapy choice and/or follow-up.

Bone marrow (BM) tissue biopsy evaluation, including trephine biopsy and clot section, is an integral part of BM investigation and is often followed by ancillary studies, in particular immunohistochemistry (IHC). IHC provides in situ coupling of morphological assessment and immunophenotype. The number of different IHC tests that can be applied to BM trephine biopsies and the number of indications for IHC testing is increasing concurrently with the development of flow cytometry and molecular diagnostic methods. An international Working Party for the Standardization of Bone Marrow IHC was formed by the International Council for Standardization in Hematology (ICSH) to prepare a set of guidelines for the standardization of BM IHC based on currently available published evidence and modern understanding of quality assurance principles as applied to IHC in general. The guidelines were discussed at the ICSH General Assemblies and reviewed by an international panel of experts to achieve further consensus and represent further development of the previously published ICSH guidelines for the standardization of BM specimens handling and reports.

Acute leukemia, myelodysplastic syndromes (MDS), myeloproliferative neoplasms and lymphomas are the most prevalent diagnoses in adults presenting with new onset cytopenia. Here, we describe two 10-color panels of surface markers (screening and comprehensive panel) applied at the Flow Cytometry Laboratory, University Health Network, Toronto, ON, Canada. A 10-color flow cytometry is applied using the stain-lyse-wash sample preparation method. In patients with

Background We have designed one-tube 14-antibody 10-color flow cytometry (FCM) panel that would provide maximum information on lymphoid and myeloid cell subsets in bone marrow aspirates (BMA) from patients with cytopenia(s). Samples and Methods BMA from 8 normal donors, from 286 non-malignant hospital controls, 92 myelodysplastic syndromes (MDS), 47 myeloproliferative neoplasms (MPN), and from 14 MDS/MPN patients were investigated. One tube 14-monoclonal antibody (MAb) 10-fluorochrome panel included: kappa+CD4 FITC, Lambda+CD8 PE, CD3-+-CD14 ECD, CD34 APC, CD20+CD56 PC7, CD10 APC-A750, CD19 APC-A700, CD33 PC5.5, CD5 PB, and CD45 KO. Kappa/lambda expression was evaluated separately in CD19+, CD10+ and CD5+ B-cells. CD4+CD3+, CD8+CD3+, CD5+CD3+ T-lymphocyte subsets were enumerated. Blasts were evaluated using CD45/SSC and CD34 gating. The FCM score for MDS (sc. Ogata score) included CD34+ myeloblast and B-progenitor cluster size, myeloblast/lymphocyte CD45 expression, and granulocyte/lymphocyte SSC ratio. Results Abnormal lymphoid populations or increased plasma cells were found in 18 patients (4%). A 43/92 BMA from MDS and 7/14 from MDS/MPN patients had score >2. Score >2 had 92.5% positive predictive value for MDS/MDS-MPN diagnosis. Negative predictive value for MDS/MDS-MPN was 83% for scores under 3 and 88% for scores under 2. All but two of normal/hospital control samples had FCM score 2 are highly indicative of MDS or MDS-MPN.

Background: Therapy directed at the central nervous system (CNS) is an essential part of the treatment for childhood acute lymphoblastic leukemia (ALL). The current evaluation of CNS involvement ba ...

Megakaryocytes are the "hallmark" of Philadelphia chromosome-negative myeloproliferative neoplasms, such as essential thrombocythemia, polycythemia vera, and primary myelofibrosis; their morphology in correlation with Janus kinase 2 (JAK2 V617F) mutation as well as clinical and laboratory parameters remains unknown.To assess the morphology of megakaryocytes in bone marrow biopsies of patients with and without JAK2 V617F mutation.Assessment of morphologic features of megakaryocytes in 112 bone marrow biopsies (52 essential thrombocythemia, 38 polycythemia vera, and 22 primary myelofibrosis) and correlation with clinical and laboratory data.JAK2 V617F mutation was detected in 24 of 52 essential thrombocythemia cases (46.2%), 36 of 38 polycythemia vera cases (97.5%), and 14 of 22 primary myelofibrosis cases (63.6%). By investigating morphometric and clinical parameters using multivariate analysis, we found that higher hemoglobin concentration, higher white blood cell counts, and lower platelet counts were significantly associated with JAK2 V617F. Striking morphologic similarities were found between polycythemia vera JAK2 V617F and primary myelofibrosis JAK2 V617F concerning the localization and cytoplasmic size of megakaryocytes. Although polycythemia vera JAK2 V617F and essential thrombocythemia JAK2 V617F shared similarities in localization, distribution, and amount of megakaryocytes, morphology was different. Megakaryocytic morphology also differed between primary myelofibrosis JAK2 V617F and essential thrombocythemia JAK2 V617F.Our results indicate that primary myelofibrosis JAK2 V617F and polycythemia vera JAK2 V617F share pathogenetic pathways, resulting in morphologically similar megakaryocytes.

Association of chronic lymphocytic leukaemia and multiple myeloma in the same individual is rare. These malignancies may be related through a common clonal origin, or exist as separate malignancies from different clones. Multiple myeloma may exist simultaneously with chronic lymphocytic leukaemia, or be diagnosed several months after chronic lymphocytic leukaemia. Common symptoms experienced in these rare cases are generally a combination of clinical features typically seen in chronic lymphocytic leukaemia and multiple myeloma separately. This report describes a 76-year-old man with coexisting chronic lymphocytic leukaemia and multiple myeloma. Diagnosis of chronic lymphocytic leukaemia preceded the diagnosis of multiple myeloma by approximately 4 years, and multiple myeloma presented with translocation t(11;14) that was not found in chronic lymphocytic leukaemia cells.

The ETV6/FLT3 fusion gene has been recently reported in association with myeloproliferative neoplasm with eosinophilia (MPN-Eo) and peripheral T cell lymphoma. Favorable clinical response to a targeted FLT3 tyrosine kinase inhibitor (FLT3 TKI) was noted. Here we report a novel phenotype associated with ETV6/FLT3 rearrangement, in a 38-year-old female with presentation of progressive lymphadenopathy. Lymph node biopsy showed mixed-phenotype acute leukemia (MPAL) with expression of T cell and myeloid markers. Bone marrow morphology was consistent with MPN-Eo and no evidence of MPAL. Karyotype analysis revealed 46, XX, t(12;13)(p13;q12). ETV6/FLT3 fusion was demonstrated by fluorescence in situ hybridization. This is the first report of ETV6/FLT3 rearrangement showing a phenotype of extramedullary T/myeloid MPAL arising in the setting of MPN-Eo. We suggest addition of this entity to the WHO category of “myeloid/lymphoid neoplasms with eosinophilia,” particularly given the possibility of clinical response to FLT3 TKI in MPAL, a disease usually associated with poor prognosis.

Genomic analysis of the clonal origin and evolution of acute promyelocytic leukemia in a unique patient with a very late (17 years) relapse

There are two broad classes (or categories) of excised human tissue: diagnostic tissue (DT) and research tissue (RT). Classification of excised human tissue does not define its ultimate use and ultimate use of excised human tissue does not define its classification. While both DT and RT can be used for research, DT has specific requirements with respect to how it must be handled if and when being accessed for research. We highlight distinguishing features of DT: (1) it is a clinical record, (2) it must be identifiable to a specific individual, (3) it is stewarded by pathology departments/clinical laboratories and (4) it has a mandatory retention period. We discuss how the further sub-classification of DT into archived DT (aDT) and excess DT (eDT) impacts the nature of its role in research. We examine the concept of DT as a clinical record and emphasize the impact of mandatory retention as it applies to how DT may be accessed for research purposes. We explain the role of post-retention eDT as a source of RT as well as procedures for access to in-retention aDT for research. Clarity of such issues will facilitate responsible access to DT for research.

Contains fulltext : 137293.pdf (Publisher’s version ) (Open Access) Del(5q) myelodysplastic syndromes defined by the International Prognostic Scoring System as low- or intermediate-1-risk (lower-risk) are considered to have an indolent course; however, recent data have identified a subgroup of these patients with more aggressive disease and poorer outcomes. Using deep sequencing technology, we previously demonstrated that 18% of patients with lower-risk del(5q) myelodysplastic syndromes carry TP53 mutated subclones rendering them at higher risk of progression. In this study, bone marrow biopsies from 85 patients treated with lenalidomide in the MDS-004 clinical trial were retrospectively assessed for p53 expression by immunohistochemistry in association with outcome. Strong p53 expression in >/= 1% of bone marrow progenitor cells, observed in 35% (30 of 85) of patients, was significantly associated with higher acute myeloid leukemia risk (P=0.0006), shorter overall survival (P=0.0175), and a lower cytogenetic response rate (P=0.009), but not with achievement or duration of 26-week transfusion independence response. In a multivariate analysis, p53-positive immunohistochemistry was the strongest independent predictor of transformation to acute myeloid leukemia (P=0.0035). Pyrosequencing analysis of laser-microdissected cells with strong p53 expression confirmed the TP53 mutation, whereas cells with moderate expression predominantly had wild-type p53. This study validates p53 immunohistochemistry as a strong and clinically useful predictive tool in patients with lower-risk del(5q) myelodysplastic syndromes. This study was based on data from the MDS 004 trial (clinicaltrials.gov identifier: NCT00179621).

Background: PCR of rearranged antigen receptor genes is the method of choice for MRD quantification in ALL. Although FCM-MRD is faster and biologically more informative than PCR, the analysis requires a high level of training. The only larger published studies using FCM-MRD based stratification (Borowitz, Blood, 2008 and 2015) showed a clear association with clinical outcome in BCP-ALL. However, MRD analyses were centralized and these studies included only one MRD-based stratification (MRD levels at the end of induction). Patients and methods: We examined FCM-MRD as stratification tool in BCP-ALL at various timepoints in a large-scale multicenter (18 MRD centers) study. A total of 1487 patients with BCP-ALL (1298 children (younger than 18 years) and 189 adults (18-45 years) are included in the study and were treated according to the NOPHO ALL2008 protocol between July 2008 and February 2016. The median follow-up time for patients in first remission was 51 months (IQR 32-75). MRD was measured by FCM and/or real time quantitative PCR on days 15, 29 (end of induction) and 79 (for standard (SR) and intermediate risk (IR) patients) and prior to and after high risk blocks. A 6-colour FCM analysis including 3 standardized antibody combinations was used and performed in 18 laboratories. Patients were stratified by FCM-MRD, or by PCR-MRD if no FCM-MRD marker was available. End-of-induction MRD (cut-off 10-3) was used to stratify patients to standard risk (SR) vs intermediate risk (IR) or IR vs high risk consolidation therapy (in case of WBC > 100 x 109/L at diagnosis). Patients with MRD >=2.5x10-1 on day 15 were stratified to high risk block therapy. Patients with MRD >=5x10-2 on day 29 or day 79/post high risk-2 block MRD >=10-3 were stratified to HSCT. Primary outcomes were 5year event-free survival (5y EFS) and 5year cumulative incidence of relapse (5y CIR). Results: Only two patients (0.14% of total) had neither an informative FCM nor a PCR marker, and an informative FCM marker combination for MRD monitoring was identified in 96.2% of patients. There was a significant correlation between FCM- and PCR-MRD levels on day 15 (r=0.77, p Based on FCM-MRD only, the median MRD level on day 15, 29 and 79/post high risk-2 block was 5x10-3, 1.1x10-4, and below detection limit, respectively. Adults had significantly higher MRD levels at all time-points (p The day 29 FCM-MRD level was closely associated with clinical outcome and a higher hazard of relapse was seen independently for a FCM-MRD >=10-3 (hazard ratio (HR) 2.4, CI 1.6-3.7, p18 year (HR 3.0, CI 1.7-5.3, p=100 (HR 2.7, CI 1.6-4.6, p=0.0001), and B-other (HR 2.1, CI 1.2-3.5, p=0.0052) or high risk B-ALL cytogenetic aberration (rearranged KMT2A/iAMPchr21/hypodiploid) (HR 3.2, CI 1.6-6.1, p=0.0006) (multivariate cause-specific Cox regression, n=1328). Patients with a day 79 FCM-MRD >=10-4 and =10-4 and =10-4 and Patients with day 15 FCM-MRD =10-3 and Conclusion: FCM-MRD performed in a multi-center setting is a clinically useful method for disease monitoring and MRD-based treatment stratification in BCP-ALL. Moreover, FCM-MRD is a reliable indicator of outcome in BCP-ALL independently of other key risk factors. Residual disease >=10-4 and Disclosures No relevant conflicts of interest to declare.

Objectives: To standardize diagnostic investigations for myeloproliferative neoplasms (MPNs) to increase homogeneity in patient care and to streamline diagnostic approaches in the most efficient and cost-effective manner. Methods: The development of Canadian expert consensus recommendations for the diagnosis of MPNs began with a review of the following: clinical evidence, daily practice, existing treatment guidelines, and availability of diagnostic tools. Each group member was assigned a specific topic, which they discussed with the entire group during several consensus meetings. Results: This document provides the Canadian MPN group’s recommendations, proposed diagnostic algorithms, and background evidence upon which decisions were made. Conclusions: Standardization of diagnostic investigations will increase homogeneity in patient care and provide a foundation for future clinical research in this rapidly evolving therapeutic area. Streamlining diagnostic approaches in the most efficient and cost-effective manner will also result in significant cost saving for the health care system.

Survival in multiple myeloma (MM) has improved significantly during recent decades both in younger and older patients.1,2 The improved survival is considered to be primarily due to new treatment options in MM, including high-dose melphalan with autologous stem cell transplantation,3 the immunomodulatory drugs and proteasome inhibitors.4,5 Recently, second malignancies have gained great clinical and scientific attention in MM as three randomized clinical trials reported an increase in second malignancies associated with lenalidomide maintenance treatment.6 In a newly published meta-analysis, exposure to lenalidomide plus oral melphalan was found to significantly increase hematologic second malignancies.7 Previously we showed that MM patients had a 26% increased risk of developing any second malignancy when compared to the general population, and an 11-fold increased risk of developing acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS).8 In the United States, second or higher-order malignancies are the third most common cancer diagnoses.9 With improved survival in MM patients, second malignancies are expected to increase in the near future and possibly contribute to problems of disease management. Importantly, it has been shown that the cumulative risk of death from MM outweighs the risk of death due to second malignancies.6 For the individual patient who develops a second malignancy, however, the outcome is of great importance. We conducted a large population-based cohort study, including all patients diagnosed with MM in Sweden, over a period of more than 50 years. This study aimed to investigate the effects of second malignancies on survival and assess changes following the introduction of modern myeloma therapy. Furthermore, as AML/MDS is over-represented in MM patients, we assessed patterns of survival specifically in these patients.

Harmonemia: a universal strategy for flow cytometry immunophenotyping—A European LeukemiaNet WP10 study

Darlington P, Haugom-Olsen H, von Sivers K, Wahlstrom J, Runold M, Svjatoha V, Porwit A, Eklund A, Grunewald J (Karolinska Institutet, Stockholm, Sweden). T-cell phenotypes in bronchoalveolar lavage fluid, blood and lymph nodes in pulmonary sarcoidosis – indication for an airborne antigen as the triggering factor in sarcoidosis. J Intern Med 2012; 272: 465–471. Background. An increased percentage of CD4+ T cells is usually observed in bronchoalveolar lavage fluid (BALF) from patients with sarcoidosis. In HLA-DRB1*03-positive patients, such T cells express the T-cell receptor (TCR) AV2S3+ gene segment. It is not known whether cells found in BALF reflect those in enlarged regional lymph nodes (LNs). Therefore, the aim of this study was to compare T-cell phenotypes in BALF, blood and mediastinal LNs. Methods. Fifteen patients underwent clinical investigation including bronchoscopy with bronchoalveolar lavage. Blood samples were drawn, and endoscopic ultrasound-guided fine-needle aspiration of enlarged mediastinal LNs was performed via the oesophagus. T cells from all three compartments were analysed by flow cytometry for markers of activity, differentiation and T regulatory function. Results. The CD4/CD8 ratio was significantly higher in BALF compared with regional LNs and was also significantly higher in LNs than in blood. The CD4+ T cells were recently activated and more differentiated in BALF than in blood and LNs. There was an accumulation of T regulatory cells (FOXP3+) in LNs and a correlation between high levels of FOXP3+ cells in BALF and in LNs. In HLA-DRB1*03-positive patients, TCR AV2S3+ CD4+ T cells were predominantly localized within BALF. Conclusions. The CD4+ T-cell phenotype in BALF indicates an active ongoing specific immune response primarily localized to the alveolar space.

BACKGROUND: The current World Health Organization classification of myelodysplastic syndromes is based morphological evaluation of bone marrow dysplasia. In clinical practice, the reproducibility of the recognition of dysplasia is usually poor especially in cases that lack specific markers such as ring sideroblasts and clonal cytogenetic abnormalities. DESIGN AND METHODS: We aimed to develop and validate a flow cytometric score for the diagnosis of myelodysplastic syndrome. Four reproducible parameters were analyzed: CD34(+) myeloblast-related and B-progenitor-related cluster size (defined by CD45 expression and side scatter characteristics CD34(+) marrow cells), myeloblast CD45 expression and granulocyte side scatter value. The study comprised a "learning cohort" (n=538) to define the score and a "validation cohort" (n=259) to confirm its diagnostic value. RESULTS: With respect to non-clonal cytopenias, patients with myelodysplastic syndrome had increased myeloblast-related cluster size, decreased B-progenitor-related cluster size, aberrant CD45 expression and reduced granulocyte side scatter (P

The 2008 edition of the WHO Classification of Tumors of Haematopoietic and Lymphoid Tissues recognizes a special category called "leukemias of ambiguous lineage." The vast majority of these rare leukemias are classified as mixed phenotype acute leukemia (MPAL), although acute undifferentiated leukemias and natural killer lymphoblastic leukemias are also included. The major immunophenotypic markers used by the WHO 2008 to determine the lineage for these proliferations are myeloperoxidase, CD19, and cytoplasmic CD3. However, extensive immunophenotyping is necessary to confirm that the cells indeed belong to 2 different lineages or coexpress differentiation antigens of more than 1 lineage. Specific subsets of MPAL are defined by chromosomal anomalies such as the t(9;22) Philadelphia chromosome BCR-ABL1 or involvement of the MLL gene on chromosome 11q23. Other MPAL are divided into B/myeloid NOS, T/myeloid NOS, B/T NOS, and B/T/myeloid NOS. MPAL are usually of dire prognosis, respond variably to chemotherapy of acute lymphoblastic or acute myeloblastic type, and benefit most from rapid allogeneic hematopoietic stem cell transplantation.

Contains fulltext : 107787.pdf (Publisher’s version ) (Open Access) BACKGROUND: The World Health Organization classification of myeloproliferative neoplasms discriminates between essential thrombocythemia and the prefibrotic phase of primary myelofibrosis. This discrimination is clinically relevant because essential thrombocythemia is associated with a favorable prognosis whereas patients with primary myelofibrosis have a higher risk of progression to myelofibrosis or blast crisis. DESIGN AND METHODS: To assess the reproducibility of the classification, six hematopathologists from five European countries re-classified 102 non-fibrotic bone marrow trephines, obtained because of sustained thrombocytosis. RESULTS: Consensus on histological classification defined as at least four identical diagnoses occurred for 63% of the samples. Inter-observer agreement showed low to moderate kappa values (0.28 to 0.57, average 0.41). The percentage of unclassifiable myeloproliferative neoplasms rose from 2% to 23% when minor criteria for primary myelofibrosis were taken into account. In contrast, the frequency of primary myelofibrosis dropped from 23% to 7%, indicating that the majority of patients with a histological diagnosis of primary myelofibrosis did not fulfill the complete criteria for this disease. Thus, over 50% of cases in this series either could not be reproducibly classified or fell into the category of unclassifiable myeloproliferative neoplasms. CONCLUSIONS: World Health Organization criteria for discrimination of essential thrombocythemia from prefibrotic primary myelofibrosis are poorly to only moderately reproducible and lead to a higher proportion of non-classifiable myeloproliferative neoplasms than histology alone. 01 maart 2012

BACKGROUND: Patients with chromosome 5 abnormalities and high-risk myelodysplastic syndromes or acute myeloid leukemia have a poor outcome. We hypothesized that increasing doses of lenalidomide may benefit this group of patients by inhibiting the tumor clone, as assessed by fluorescence in situ hybridization for del(5q31).DESIGN AND METHODS: Twenty-eight patients at diagnosis or with relapsed disease and not eligible for standard therapy (16 with acute myeloid leukemia, 12 with intermediate-risk 2 or high-risk myelodysplastic syndrome) were enrolled in this prospective phase II multicenter trial and treated with lenalidomide up to 30 mg daily for 16 weeks. Three patients had isolated del(5q), six had del(5q) plus one additional aberration, 14 had del(5q) and a complex karyotype, four had monosomy 5, and one had del(5q) identified by fluorescence in situ hybridization only.RESULTS: Major and minor cytogenetic responses, assessed by fluorescence in situ hybridization, were achieved in 5/26 (19%) and 2/26 (8%) patients, respectively, who received one or more dose of lenalidomide, while two patients achieved only a bone marrow response. Nine of all 26 patients (35%) and nine of the ten who completed the 16 weeks of trial responded to treatment. Using the International Working Group criteria for acute myeloid leukemia and myelodysplastic syndrome the overall response rate in treated patients with acute myeloid leukemia was 20% (3/15), while that for patients with myelodysplastic syndrome was 36% (4/11). Seven patients stopped therapy due to progressive disease and nine because of complications, most of which were disease-related. Response rates were similar in patients with isolated del(5q) and in those with additional aberrations. Interestingly, patients with TP53 mutations responded less well than those without mutations (2/13 versus 5/9, respectively; P=0.047). No responses were observed among 11 cases with deleterious TP53 mutations.CONCLUSIONS: Our data support a role for higher doses of lenalidomide in poor prognosis patients with myelodysplastic syndrome and acute myeloid leukemia with deletion 5q. (Clinicaltrials.gov identifier NCT00761449).

On average, 30% of patients with myelodysplastic syndrome (MDS) develop overt acute myeloid leukemia (AML) during the course of the disease. There is a continuous search for the best model of individual risk assessment for MDS patients. In this review, we summarize current findings on factors that have been associated with increased risk of AML transformation. These include laboratory values such as high lactate dehydrogenase levels, complex karyotypes, numbers and aberrant immunophenotype of bone marrow blasts, bone marrow-related features such as numbers and distribution of CD34+ cells, and recently established molecular markers. A wide range of described molecular aberrations in MDS, including various gene mutations, chromosomal instability, short telomeres, high levels of gene methylation, and histone modification, partly explains clinical heterogeneity of this disease. Continuous research will bring more insight in the pathogenesis of various MDS categories, making individual risk assessment and tailored therapy for each patient possible.

Purpose To determine the frequency of TP53 mutations and the level of p53 protein expression by immunohistochemistry (IHC) in low-risk myelodysplastic syndromes (MDS) with del(5q) and to assess their impact on disease progression. Patients and Methods Pre- and postprogression bone marrow (BM) samples from 55 consecutive patients with International Prognostic Scoring System low risk (n = 32) or intermediate-1 risk (n = 23) were studied by next-generation sequencing of TP53. IHC for p53 was performed on 148 sequential BM samples. Results TP53 mutations with a median clone size of 11% (range, 1% to 54%) were detected in 10 patients (18%) already at an early phase of the disease. Mutations were equally common in low-risk and intermediate-1–risk patients and were associated with evolution to acute myeloid leukemia (5 of 10 v 7 of 45; P = .045). Nine of 10 patients carrying mutations showed more than 2% BM progenitors with strong p53 staining. The probability of a complete cytogenetic response to lenalidomide was lower in mutated patients (0 of 7 v 12 of 24; P = .024). Conclusion By using sensitive deep-sequencing technology, we demonstrated that TP53 mutated populations may occur at an early disease stage in almost a fifth of low-risk MDS patients with del(5q). Importantly, mutations were present years before disease progression and were associated with an increased risk of leukemic evolution. TP53 mutations could not be predicted by common clinical features but were associated with p53 overexpression. Our findings indicate a previously unrecognized heterogeneity of the disease which may significantly affect clinical decision making.

Leukemic cells from 230 children with newly diagnosed B-cell precursor ALL were tested for in vitro drug resistance to a panel of anti-cancer drugs. Minimal residual disease (MRD) was measured by RQ-PCR. During follow-up, 24 relapses occurred in the 159 children with MRD

The features of 100 mixed-phenotype acute leukemias (MPALs), fulfilling WHO 2008 criteria, are documented. Myeloid and T-lineage features were demonstrated by cytoplasmic myeloperoxidase and CD3; B-lineage features were demonstrated by at least 2 B-lymphoid markers. There were 62 men and 38 women; 68% were adults. Morphology was consistent with acute lymphoblastic leukemia (ALL; 43%), acute myeloid leukemia (AML; 42%), or inconclusive (15%). Immunophenotyping disclosed B + myeloid (59%), T + myeloid (35%), B + T (4%), or trilineage (2%) combinations. Cytogenetics evidenced t(9;22)/(Ph+) (20%), 11q23/ MLL rearrangements (8%), complex (32%), aberrant (27%), or normal (13%) karyotypes. There was no correlation between age, morphology, immunophenotype, or cytogenetics. Response to treatment and outcome were available for 67 and 70 patients, respectively; 27 received ALL, 34 AML, 5 a combination of ALL + AML therapy, and 1 imatinib. ALL treatment induced a response in 85%, AML therapy in 41%; 3 of 5 patients responded to the combination therapy. Forty (58%) patients died, 33 of resistant disease. Overall median survival was 18 months and 37% of patients are alive at 5 years. Age, Ph+, and AML therapy were predictors for poor outcome (P < .001; P = .002; P = .003). MPAL is confirmed to be a poor-risk disease. Adults and Ph+ patients should be considered for transplantation in first remission.

The European LeukemiaNet (ELN), workpackage 10 (WP10) was designed to deal with diagnosis matters using morphology and immunophenotyping. This group aimed at establishing a consensus on the required reagents for proper immunophenotyping of acute leukemia and lymphoproliferative disorders. Animated discussions within WP10, together with the application of the Delphi method of proposals circulation, quickly led to post-consensual immunophenotyping panels for disorders on the ELN website. In this report, we established a comprehensive description of these panels, both mandatory and complementary, for both types of clinical conditions. The reason for using each marker, sustained by relevant literature information, is provided in detail. With the constant development of immunophenotyping techniques in flow cytometry and related software, this work aims at providing useful guidelines to perform the most pertinent exploration at diagnosis and for follow-up, with the best cost benefit in diseases, the treatment of which has a strong impact on health systems.

Minimal residual disease (MRD) assessment is a powerful prognostic factor for determining the risk of relapse in childhood acute lymphoblastic leukaemia (ALL). In this Swedish multi-centre study of childhood ALL diagnosed between 2002 and 2006, the MRD levels were analysed in 726 follow-up samples in 228 children using real-time quantitative polymerase chain reaction (RQ-PCR) of rearranged immunoglobulin/T-cell receptor genes and multicolour flow cytometry (FCM). Using an MRD threshold of 0·1%, which was the sensitivity level reached in all analyses, the concordance between RQ-PCR and FCM MRD values at day 29 was 84%. In B-cell precursor ALL, an MRD level of ≥0·1% at day 29 predicted a higher risk of bone marrow relapse (BMR) with both methods, although FCM was a better discriminator. However, considering the higher median MRD values achieved with RQ-PCR, a higher MRD cut-off (≥0·2%) improved the predictive capacity of RQ-PCR. In T-ALL, RQ-PCR was notably superior to FCM in predicting risk of BMR. That notwithstanding, MRD levels of ≥0·1%, detected by either method at day 29, could not predict isolated extramedullary relapse. In conclusion, the concordance between RQ-PCR and FCM was high and hence both methods are valuable clinical tools for identifying childhood ALL cases with increased risk of BMR.

Risk factors for acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) in patients with multiple myeloma (MM) are not well understood. We have previously shown that monoclonal gammopathy of undetermined significance (MGUS) is associated with an excess risk of AML/MDS, indicating an inherent excess risk of AML/MDS in plasma cell disorders. In addition, randomized studies have shown that lenalidomide maintenance is associated with increased relative risk of AML/MDS. The excess risk of AML/MDS in lenalidomide treated patients has been found to be most prominent in patients that also receive alkylating agents. A recent randomized clinical study shows that patients undergoing delayed autologous transplant have the same overall survival as patients undergoing upfront autologous transplant with high dose melphalan at 3 years of follow-up. Also emerging data shows that patients with minimal residual disease (MRD) negativity have very similar progression free survival independent of therapy. Thus, patients treated with modern combination therapy who obtain MRD negativity might not have additional benefit from upfront autologous transplant. We were motivated to better define the relationship of alkylating therapy to risk of secondary malignancies. Information was obtained regarding all subsequent hematologic malignancies in all patients diagnosed with MM from January 1, 1958 to December 31, 2011 identified from the Swedish Cancer Registry. Each patient with MM and a subsequent AML/MDS diagnosis (i.e., cases) was matched to another MM patient without AML/MDS for year of birth, sex, and date of MM diagnosis (i.e., control group). Detailed information on baseline characteristics at diagnosis and treatment was collected from medical records. Characteristics at MM diagnosis, radiation therapy, and high dose melphalan with autologous stem cell transplant (ASCT) were compared between groups with Kruskal-Wallis and Chi-square tests. Cumulative chemotherapy doses were analyzed with one way ANOVA. Post hoc analysis with Fisher´s least significant difference (LSD) was performed. A total of 26,627 patients were diagnosed with MM in Sweden during the study period. Of these, 113 patients developed subsequent AML/MDS. Data was found for 87 and they were matched to 69 controls. The demographic and clinical characteristics for both groups are listed in Table. The median age at MM diagnosis was 73 years (38 females and 49 males) for cases and 70 years (31 females and 38 males) for controls. The median time from MM diagnosis to AML/MDS diagnosis was 3.8 years (IQR 2.8 - 5.8). The mean cumulative melphalan dose was significantly higher (1,321 mg , SD ± 1,165 mg) for cases, than for the controls (709 mg , SD ± 565 mg)(p= In this large nationwide population based study including almost 27,000 MM patients diagnosed during over >50 years period in Sweden, we confirm our previous preliminary findings that higher mean cumulative dose of melphalan was associated with increased risk of developing AML/MDS. With the average myeloma patient living over 10-15 years from diagnosis, strategies to avoid secondary complications is becoming more important. Our results showing that melphalan is associated with an increased risk of AML/MDS, call for studies using response driven (i.e. MRD based) therapy in myeloma; and high dose melphalan is rather offered to patients who are MRD positive, and/or as relapse therapy. Disclosures Landgren: Pfizer: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees.

Objectives: Patients with near-tetraploid (karyotype: 81 - 103 chromosomes) acute lymphoblastic leukemia (NT-ALL) constitute about 1% of childhood ALL and data reported on them are limited and controversial. The aim of the study was to enlarge the knowledge on these rarely occurring ALL. Methods: The members of the European Group for Immunophenotyping of Leukemias (EGIL) searched retrospectively their databases for NT-ALL patients. Results: We collected data of 36 European children from seven European countries with NT-ALL diagnosed since 1992. All patients reached complete remission (CR) after induction chemotherapy. Their blasts were negative for peroxidase and BCR-ABL1. Ten children were diagnosed as T-cell ALL (T-ALL) EGIL categories (T-I n = 2, T-II n = 2, T-III n = 3, T-IV n = 3) and four displayed various structural chromosomal abnormalities. Eight of 10 T-ALL remained in 1st CR; one died in CR from sepsis and one is alive in 2nd CR. Median survival was 88 (7-213) months. B-cell precursor (BCP) ALL was diagnosed in 26 children. Thirteen were positive for ETV6-RUNX1 and are alive in 1st CR for 32-147 months. Ten children were ETV6-RUNX1 negative and remained in 1st CR for 16-163 months. One girl with hypodiploid and NT metaphases and ETV6-RUNX1-negative BCP-ALL and one of two boys with NT-BCP-ALL not examined for ETV6-RUNX1 died of infection after stem cell transplantation in 2nd/3rd CR. Secondary myelodysplastic syndrome developed in two patients with NT-BCP-ALL. Conclusions: Our data demonstrate immunophenotypic, cytogenetic, and molecular heterogeneity of NT-ALL and favorable prognosis of most NT-ALL across different immunophenotypic and/or genetic ALL subtypes.

This prospective Phase II study is the first to assess the feasibility and efficacy of maintenance 5-azacytidine for older patients with high-risk myelodysplastic syndrome (MDS), chronic myelomonocytic leukaemia and MDS-acute myeloid leukaemia syndromes in complete remission (CR) after induction chemotherapy. Sixty patients were enrolled and treated by standard induction chemotherapy. Patients that reached CR started maintenance therapy with subcutaneous azacytidine, 5/28 d until relapse. Promoter-methylation status of CDKN2B (P15 ink4b), CDH1 and HIC1 was examined pre-induction, in CR and 6, 12 and 24 months post CR. Twenty-four (40%) patients achieved CR after induction chemotherapy and 23 started maintenance treatment with azacytidine. Median CR duration was 13.5 months, >24 months in 17% of the patients, and 18-30.5 months in the four patients with trisomy 8. CR duration was not associated with CDKN2B methylation status or karyotype. Median overall survival was 20 months. Hypermethylation of CDH1 was significantly associated with low CR rate, early relapse, and short overall survival (P = 0.003). 5-azacytidine treatment, at a dose of 60 mg/m(2) was well tolerated. Grade III-IV thrombocytopenia and neutropenia occurred after 9.5 and 30% of the cycles, respectively, while haemoglobin levels increased during treatment. 5-azacytidine treatment is safe, feasible and may be of benefit in a subset of patients.

Tumor necrosis factor (TNF) and interleukin 10 (IL10) are promising candidate susceptibility genes for non-Hodgkin lymphoma (NHL). Chromosomal translocation breakpoint genes are of interest, given their documented involvement in lymphoma progression.We analyzed 11 polymorphisms in BCL2, CCND1, MYC, TNF, and IL10 in a large, population-based, Danish-Swedish case-control study including 2,449 NHL cases and 1,980 controls. Relative risk of NHL was computed as odds ratios (OR).There was no clear evidence of associations between variants in BCL2, CCND1, and MYC and risk of NHL overall or subtypes. TNF rs1800629 was associated with risk of NHL (OR 1.53, 95% confidence interval, CI, 1.06-2.19 for minor allele homozygosity), T-cell lymphoma (OR 2.54, CI 1.27-5.09) and mantle cell lymphoma (OR 2.84, CI 1.38-5.87). IL10 rs1800890 was associated with risk of diffuse large B-cell lymphoma (OR 1.41, CI 1.08-1.85 for minor allele homozygosity) and mantle cell lymphoma (OR 1.77, CI 1.04-3.00). We did not replicate a previously reported interaction with autoimmunity.We found no support for a role of the studied variants in BCL2, CCND1, or MYC in risk of NHL or subtypes, but we provide further evidence of putative susceptibility loci in TNF and IL10 for specific NHL subtypes.

The issue of angiogenesis and its clinical relevance in childhood acute lymphoblastic leukaemia (ALL) is controversial. In the present study, microvessel density (MVD), analysed in 185 diagnostic bone marrow biopsies, was higher in T-cell ALL compared to B-cell precursor (BCP)-ALL (P = 0.013). In the BCP group, cases with t(12;21) were characterized by a low MVD while patients with high-hyperdiploid leukaemia (HeH, 51-61 chromosomes) showed a high MVD compared to other BCP patients (P = 0.001 and 0.002 respectively). There was a correlation between MVD and white blood cell (WBC) count in high-risk BCP patients (P = 0.021). In addition, BCP patients with a high marrow reticulin fibre density and high MVD had an unfavourable outcome compared to the other BCP patients (P = 0.002). The fraction of vessels in which lumina were filled with blasts (blast congested vessel fraction) correlated strongly with WBC count (P0.001). These findings indicate that the angiogenic process interacts with other stroma-factors, such as reticulin fibre density, in its effect on outcome, and is coupled to both the ALL genotype and phenotype. One possible implication is that different subtypes of childhood ALL may respond differently to anti-angiogenic drugs as a supplement in first-line treatment.

Recently the World Health Organization (WHO), in collaboration with the European Association for Haematopathology and the Society for Hematopathology, published a revised and updated edition of the WHO Classification of Tumors of the Hematopoietic and Lymphoid Tissues. The 4th edition of the WHO classification incorporates new information that has emerged from scientific and clinical studies in the interval since the publication of the 3rd edition in 2001, and includes new criteria for the recognition of some previously described neoplasms as well as clarification and refinement of the defining criteria for others. It also adds entities—some defined principally by genetic features—that have only recently been characterized. In this paper, the classification of myeloid neoplasms and acute leukemia is highlighted with the aim of familiarizing hematologists, clinical scientists, and hematopathologists not only with the major changes in the classification but also with the rationale for those changes.